An essential step in the replication cycle of retroviruses is integration of a DNA copy of the viral genome, made by reverse transcription of viral RNA, into the chromosome of a newly infected cell. The objective of this project is to analyze the molecular mechanism of this integration step and to develop a simple in vitro assay system that may be used to efficiently screen for chemical inhibitors of this step in the viral life cycle. Since there is no known host analogue of this integration reaction, it is hoped that some of these inhibitors will prove to be clinically useful as antiviral drugs against HIV. We currently use Moloney murine leukemia virus (MoMLV) as a model system for studying the mechanism of retroviral DNA integration. Utilizing a cell-free reaction system, in which extracts of virus- infected cells provide both the viral DNA substrate and protein factors required for integration, we have analyzed the structure reveals that MoMLv integrates by a mechanism that is strikingly similar to the transposition mechanism of a prokaryotic transposon, suggesting that this reaction is highly conserved among many mobile genetic elements. We have recently developed a cell-free reaction system for integration of exogenously added viral DNA. This system is being exploited to determine which viral polypeptides are required for the integration reaction. The feasibility of screening for inhibitors of integration by means of the cell-free reaction with endogenous viral DNA has already been demonstrated on a laboratory scale.